Blood, 15 June 2002, Vol. 99, No. 12, pp. 4644-4646
CORRESPONDENCE
To the editor:
Effects of adenosine 5'-diphosphate (ADP)
receptor blockade on platelet aggregation under flow
We read with interest the article presented by Dr Turner and
colleagues demonstrating the effects of P2Y1 and
P2Y12 adenosine 5'-diphosphate (ADP) receptor
blockade on platelet aggregation and thrombus formation under flow
conditions.1 However, we have serious concerns about their
statement, "blockade of P2Y12 alone or blockade of
P2Y1 alone did not reduce thrombus formation on
VWF-collagen surface," not only because these results are not in
agreement with previously published findings using prodrugs of
thienopyridine antiplatelet agents2 that inhibit the
P2Y12 receptor, but also because they are not in agreement
with our own experimental results (Figure
1). We used a similar flow chamber system
equipped with an epi-fluorescent videomicroscope3 and perfused whole blood containing platelets rendered fluorescent by the
addition of mepacrine on immobilized bovine type I collagen, in a
manner similar to that described by Turner et al1 and at a
slightly lower shear rate of 1500 s-1. However, our
results, shown in Figure 1, clearly demonstrate that AR-C69931MX alone
at the dose of 500 nM was sufficient to inhibit platelet thrombus
formation on the surface of immobilized collagen. We used the specific
and reversible antithrombin agent argatroban instead of heparin as the
anticoagulant, to exclude the possible pleiotropic effects of
heparin.3,4 Also, we perfused the blood on the collagen
surface for 5 minutes instead of for 1 minute. We believe that the
second of the aforementioned differences explains the differences in
our results, because the inhibitory effects of AR-C69931MX were not
clear at 1 minute even in our study (Figure 1). Thus, we believe that
the authors should have extended the perfusion period further in their
experiments before concluding that "blockade of P2Y12
alone" "did not reduce thrombus formation on VWF-collagen."
Indeed, numerous clinical studies have clearly demonstrated the
effectiveness of thienopyridine antiplatelet agents that block the
P2Y12 receptor in preventing conditions associated with
arterial thrombosis, such as acute myocardial infarction,5
in which VWF-mediated platelet thrombus formation plays an important
role.6 Therefore, we believe that P2Y12
inhibition by itself is sufficient to inhibit platelet thrombus formation at sites of endothelial damage exposed to arterial levels of
shear stress.
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| Figure 1.
Effects of AR-C69931MX on platelet thrombus formation on
the surface of collagen under flow conditions.
Blood containing platelets rendered fluorescent by the addition of
mepacrine and anticoagulated with the specific antithrombin agent
argatroban was perfused on collagen-coated glass coverslips at a wall
shear rate of 1500 s-1 for 5 minutes, either in the absence
(Control) or presence of the specific P2Y12 antagonist
AR-C69931MX at a final concentration of 500 nM. Platelet thrombi formed
on the collagen surface were detected by inverted-stage epi-fluorescent
videomicroscopy (DM IRB, 1RB-FLUO, Leica, Germany), as demonstrated in
the lower panel of the figure. The microscopic images were digitized
online with a photosensitive CCD camera (L-600, Leica, Germany).
Surface coverage by the platelets was calculated using National
Institutes of Health (NIH) Image software (public domain software by Dr
Wayne Rasband, NIH, Bethesda, MD, version 1.62), and the results are
shown in the upper panel of the figure. These results are the mean ± SD of the 3 experiments, while the results shown in the lower panel
show 1 representative result of the 3 experiments.
|
|
Shinya Goto, Noriko Tamura, and Shunnosuke Handa
Correspondence: Shinya Goto, Division of Cardiology, Tokai
University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa
259-1193, Japan; e-mail: shinichi{at}is.icc.u-tokai.ac.jp
Acknowledgments
We acknowledge the financial support from a Grant-in-Aid for
Scientific Research in Japan 13670744; grant JSPS-RFTF97I00201 from the
Japanese Society for the Promotion of Science; and a grant from the
Science Frontier Program of MESSC of Japan.
References
1.
Turner NA, Moake JL, McIntire LV.
Blockade of adenosine diphosphate receptors P2Y12 and P2Y1 is required to inhibit platelet aggregation in whole blood under flow.
Blood.
2001;98:3340-3345[Abstract/Free Full Text].
2.
Roald HE, Barstad RM, Kierulf P, et al.
Clopidogrel: a platelet inhibitor which inhibits thrombogenesis in non-anticoagulated human blood independently of the blood flow conditions.
Thromb Haemost.
1994;71:655-662[Medline]
[Order article via Infotrieve].
3.
Eto K, Goto S, Shimazaki T, et al.
Two distinct mechanisms are involved in stent thrombosis under flow conditions.
Platelets.
2001;12:228-235[CrossRef][Medline]
[Order article via Infotrieve].
4.
Tsuji S, Sugimoto M, Miyata S, Kuwahara M, Kinoshita S, Yoshioka A.
Real-time analysis of mural thrombus formation in various platelet aggregation disorders: distinct shear-dependent roles of platelet receptors and adhesive proteins under flow.
Blood.
1999;94:968-975[Abstract/Free Full Text].
5.
CAPRIE Steering Committee.
A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE).
Lancet.
1996;348:1329-1339[CrossRef][Medline]
[Order article via Infotrieve].
6.
Goto S, Sakai H, Goto M, et al.
Enhanced shear-induced platelet aggregation in acute myocardial infarction.
Circulation.
1999;99:608-613[Abstract/Free Full Text].
Response:
Blockade of adenosine diphosphate receptors P2Y12
and P2Y1 is required to inhibit platelet aggregation in
whole blood under flow
The Letter to the Editor by Goto et al contains some potentially
interesting data but also some generally misleading comments.
We found that ARMX alone did not inhibit platelet thrombus formation
onto the collagen surface at 3000 s
1 after 1 minute of
flow.1 Goto et al also show in the upper portion of their
Figure 1 that after 1 minute of flow at 1500 s1 there is
no difference in the platelet surface coverage between control samples
and ARMX addition. Only at longer periods of flow (3 or 5 minutes) did
Goto et al find that ARMX reduced the percent of the collagen surface
covered by platelets (by about 25% to 30% at 1500 s1).
They also fail to mention the striking inhibition of platelet deposition seen after 1 minute of perfusion when both P2Y12
and P2Y1 inhibitors are present1(figs5-7). No
data on dual inhibition are presented in the letter of Goto et al.
Goto et al fail to mention that we reported in our paper that ARMX
alone inhibited shear-induced platelet aggregation by more than 50% at
both 750 and 1500 s1 for 30 seconds in the
viscometer1(fig1). This inhibition was accentuated by
concurrent addition of A3P5P. There are no results presented on bulk
shear (viscometry studies) in the letter by Goto et al.
In summary, we believe the limited data presented by Goto et al are
consistent with our data1 at the same time points of perfusion, even though the shear rates differ by a factor of 2. The
relatively small differences at longer time points with just the
P2Y12 inhibition may be true, but the magnitude of change in platelet deposition is actually not impressive compared to what is
seen with blockade of both P2Y12 and P2Y1
receptors concurrently. Data on limited effects of each inhibitor
separately in bulk shear stress (viscometry) studies were already
presented in our earlier paper.1
Nancy A.Turner, Joel L. Moake and Larry V. McIntire
Correspondence: Larry V. McIntire, Dept of Bioengineering, Rice
University, 6100 Main St, Houston, TX 77005; e-mail:
mcintire{at}rice.edu
References
1.
Turner N, Moake J, McIntire L.
Blockade of adenosine diphosphate receptors P2Y12 and P2Y1 is required to inhibit platelet aggregation in whole blood under flow.
Blood.
2001;98:3340-3345[Abstract/Free Full Text].
